Comparative physiological and genomic characterization of a novel <i>Nitrobacter vulgaris</i> strain from a nitrate-contaminated subsurface

Nitrite-oxidizing bacteria (NOB) represent a crucial node in the global nitrogen cycle. By catalyzing the second step of nitrification-the oxidation of nitrite to nitrate to generate energy for growth-NOB activity controls the fate of nitrite (NO₂^-) in aerobic environments. Despite thriving in diverse environments, including soils, freshwater, marine ecosystems, subsurface habitats, and water treatment systems, organisms capable of nitrite oxidation are confined to <i>Nitrobacter, Nitrospira, Nitrospina, Nitrotoga,</i> and a few other specific lineages. The genus <i>Nitrobacter</i>, recognized for its facultative heterotrophic metabolism, is often associated with high-nitrogen environments. Here, we report the physiological characterization of a novel strain, <i>Nitrobacter vulgaris</i> strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface. Growth inhibition experiments revealed that strain MLSD-S22 and the <i>N. vulgaris</i> type strain Z exhibited similar sensitivities to nitrite and nitrate, with nitrite being the most inhibitory. Microrespirometry demonstrated that the two <i>N. vulgaris</i> strains and <i>Nitrobacter winogradskyi</i> Nb-255 possessed higher affinities for nitrite and oxygen than previously reported for <i>Nitrobacter</i>, suggesting potential to compete in low-substrate environments. Long-read DNA sequencing provided a complete genome for strain MLSD-S22, revealing two plasmids and an intact nitrous oxide (N₂O) reduction operon-an unexpected feature for <i>Nitrobacter</i>. While N₂O reduction activity was not observed under the tested conditions, this discovery raises questions about the contribution of <i>Nitrobacter</i> NOB to the N₂O sink. These findings broaden the physiological and genomic diversity of <i>Nitrobacter</i>, offering new insights into their adaptation strategies and providing a framework for future evaluation of their potential roles in nitrogen loss.IMPORTANCE<i>Nitrobacter</i> species play a key role in nitrogen cycling; however, their physiological adaptations and genomic diversity remain largely underexplored. This study characterizes <i>Nitrobacter vulgaris</i> strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface, revealing unexpected genomic and metabolic traits. Higher nitrite and oxygen affinities compared to previous reports indicate that some <i>Nitrobacter</i> are more competitive in low-substrate environments than previously believed. The presence of a complete nitrous oxide (N₂O) reduction operon highlights the genomic potential related to N₂O reduction within this genus; however, we did not observe N₂O reduction under the conditions tested. By expanding <i>Nitrobacter</i> ecophysiology, this work provides a foundation for future research on the metabolic flexibility of <i>Nitrobacter</i> and their contributions to nitrogen transformations in diverse environments.